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SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins
Xiu-Jun Yu1, Javier Ruiz-Albert, Kate E Unsworth
1Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College School of Medicine, Armstrong Road, London SW7 2AZ, UK.
Abstract:
Replication of Salmonella typhimurium in host cells depends in part on the action of the Salmonella Pathogenicity Island 2 (SPI-2) type III secretion system (TTSS), which translocates bacterial effector proteins across the membrane of the Salmonella-containing vacuole (SCV). We have shown previously that one activity of the SPI-2 TTSS is the assembly of a coat of F-actin in the vicinity of bacterial microcolonies. To identify proteins involved in SPI-2 dependent actin polymerization, we tested strains carrying mutations in each of several genes whose products are proposed to be secreted through the SPI-2 TTSS, for their ability to assemble F-actin around intracellular bacteria. We found that strains carrying mutations in either sseB, sseC, sseD or spiC were deficient in actin assembly. The phenotypes of the sseB-, sseC- and sseD- mutants can be attributed to their requirement for translocation of SPI-2 effectors. SpiC was investigated further in view of its proposed role as an effector. Transient expression of a myc::SpiC fusion protein in Hela cells did not induce any significant alterations to the host cell cytoskeleton, and failed to restore actin polymerization around intracellular spiC- mutant bacteria. However, the same protein did complement the mutant phenotype when expressed from a plasmid within bacteria. Furthermore, spiC was found to be required for SPI-2 mediated secretion of SseB, SseC and SseD in vitro. An antibody against SpiC detected the protein on immunoblots from total cell lysates of S. typhimurium expressing SpiC from a plasmid, but it was not detected in secreted fractions after exposure of cells to conditions that result in secretion of other SPI-2 effector proteins. Investigation of the trafficking of SCVs containing a spiC- mutant in macrophages revealed only a low level of association with the lysosomal marker cathepsin D, similar to that of wild-type bacteria. Together, these results show that SpiC is involved in the process of SPI-2 secretion and indicate that phenotypes associated with a spiC- mutant are caused by the inability of this strain to translocate effector proteins, thus calling for further investigation into the function(s) of this protein.
Insights
Salmonella typhimurium replication relies on the SPI-2 type III secretion system (TTSS). Mutations in sseB, sseC, sseD, or spiC disrupt F-actin assembly around bacteria, indicating SpiC
Area of Science:
- Microbiology
- Cell Biology
- Bacterial Pathogenesis
Background:
- Salmonella typhimurium replication within host cells is facilitated by the Salmonella Pathogenicity Island 2 (SPI-2) type III secretion system (TTSS).
- The SPI-2 TTSS translocates bacterial effector proteins into the Salmonella-containing vacuole (SCV), influencing host cell processes.
- Previous studies indicated that the SPI-2 TTSS is involved in assembling F-actin around intracellular bacterial microcolonies.
Purpose of the Study:
- To identify proteins essential for SPI-2 dependent F-actin polymerization around intracellular Salmonella.
- To elucidate the role of SpiC in the SPI-2 secretion pathway and its contribution to bacterial replication.
Main Methods:
- Screening of Salmonella typhimurium strains with mutations in putative SPI-2 secreted effector genes for defects in F-actin assembly.
- Complementation assays using a myc::SpiC fusion protein expressed intracellularly or within bacteria.
- In vitro secretion assays to assess the translocation of SPI-2 effectors (SseB, SseC, SseD) in wild-type and mutant strains.
- Immunoblotting to detect SpiC in bacterial lysates and secreted fractions.
- Analysis of Salmonella-containing vacuole (SCV) trafficking using cathepsin D as a lysosomal marker in macrophages.
Main Results:
- Mutations in sseB, sseC, sseD, and spiC genes resulted in a deficiency in F-actin assembly around intracellular Salmonella.
- SpiC was found to be crucial for the SPI-2 mediated secretion of SseB, SseC, and SseD in vitro.
- SpiC's function was dependent on its expression within the bacteria, not as a translocated effector protein affecting the host cytoskeleton.
- SCVs containing spiC mutants showed limited association with lysosomes, similar to wild-type bacteria.
Conclusions:
- SpiC plays a critical role in the SPI-2 secretion pathway, likely facilitating the translocation of effector proteins.
- The observed F-actin assembly defect in spiC mutants is a consequence of impaired effector protein secretion.
- Further research is warranted to fully define the specific functions of SpiC within the SPI-2 secretion mechanism.